Increasing evidence has identified the formation of advanced glycation end products (AGEs) as a major pathogenic link between hyperglycemia and diabetes-related complications. As the precursor of AGEs, methylglyoxal (MGO) is observed at relative higher plasma levels (2–6 fold) in diabetic patients than in healthy subjects and are also found in many food products, beverages and cigarette smoke. Chronic intake of exogenous MGO has been demonstrated as one of the cause factors for the development of diabetes. Therefore, decreasing the levels of MGO will be an effective approach to reduce the formation of AGEs and the development of diabetic complications. In this study, we investigated whether long-term treatment of MGO in drinking water can cause protein glycation in mice and whether dietary flavonoids, such as quercetin, can prevent the formation of AGEs via trapping MGO. Our results indicate that chronic MGO administration (0.06% in drinking water for 20 weeks) significantly elevates the levels of MGO and AGEs in mouse plasma, liver and kidney, and cause liver injury. Dietary quercetin treatment (0.067% and 0.2% in diet) ameliorates MGO-induced liver injury and prevents the formation of AGEs at least in Kidney. Further mechanistic studies demonstrate that both quercein and its major metabolite isorhamnetin could trap MGO in mice to form mono- and di-MGO adducts. In addition, quercetin could increase glyoxalase I and II expression and GSH/GSSG ratio to promote the detoxification of MGO.